Affiliation: MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, United Kingdom.

ABSTRACTRecent studies have established that masculinization of the male reproductive tract is programmed by androgens in a critical fetal 'masculinization programming window' (MPW). What is peculiar to androgen action during this period is, however, unknown. Studies from 20 years ago in mice implicated prostaglandin (PG)-mediation of androgen-induced masculinization, but this has never been followed up. We therefore investigated if PGs might mediate androgen effects in the MPW by exposing pregnant rats to indomethacin (which blocks PG production by inhibiting cyclooxygenase activity) during this period and then examining if androgen production or action (masculinization) was affected. Pregnant rats were treated with indomethacin (0.8 mg/kg/day; e15.5-e18.5) to encompass the MPW. Indomethacin exposure decreased fetal bodyweight (e21.5), testis weight (e21.5) and testicular PGE2 (e17.5, e21.5), but had no effect on intratesticular testosterone (ITT; e17.5) or anogenital index (AGI; e21.5). Postnatally, AGI, testis weight and blood testosterone were unaffected by indomethacin exposure and no cryptorchidism or hypospadias occurred. Penis length was normal in indomethacin-exposed animals at Pnd25 but was reduced by 26% (p<0.001) in adulthood, an effect that is unexplained. Our results demonstrate that indomethacin can effectively decrease intra-testicular PGE2 level. However, the resulting male phenotype does not support a role for PGs in mediating androgen-induced masculinization during the MPW in rats. The contrast with previous mouse studies is unexplained but may reflect a species difference.

pone-0062556-g001: Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day) on (A) testicular PGE2 levels at e17.5 and (B) intra-testicular testosterone content at e17.5.Values are means ± SEM for N = 5 for the top graph and N = 7–28 for the bottom graph, from a minimum of 3 litters. **p<0.01, in comparison with respective control.

Mentions:
At 4 h after maternal indomethacin treatment, PGE2 levels in the fetal testis were significantly decreased at e17.5 (during the MPW) when compared to controls (Fig. 1A), indicating effective exposure of the fetus. To provide further reassurance on this, we established separately that our indomethacin treatment protocol suppressed PGE2 levels in the fetal testis on e21.5 (Fig S1). However, fetal exposure to indomethacin during the MPW did not significantly alter intratesticular testosterone (ITT) levels at e17.5, in comparison to vehicle exposed animals (Fig 1B). Testosterone levels/androgen action during the MPW is reflected in AGD/AGI measurements from e21.5 onwards [4],[5]. In keeping with the data for e17.5 ITT, fetal indomethacin exposure during the MPW did not affect AGI at e21.5 (Fig. 2A), but did decrease fetal body weight of e21.5 male fetuses (Fig. 2B). Furthermore, exposure to indomethacin caused a significant decrease (27%) in fetal testis weight at e21.5 and this decrease occurred despite the fact that indomethacin treatment had ceased after e18.5 (Fig. 2C). If indomethacin treatment (0.8 mg.kg/day) was administered for a longer period (e15.5–e20.5), a similar decrease was found in testis weight (data not shown).

pone-0062556-g001: Effects of maternal exposure to vehicle or indomethacin (0.8 mg/kg/day) on (A) testicular PGE2 levels at e17.5 and (B) intra-testicular testosterone content at e17.5.Values are means ± SEM for N = 5 for the top graph and N = 7–28 for the bottom graph, from a minimum of 3 litters. **p<0.01, in comparison with respective control.

Mentions:
At 4 h after maternal indomethacin treatment, PGE2 levels in the fetal testis were significantly decreased at e17.5 (during the MPW) when compared to controls (Fig. 1A), indicating effective exposure of the fetus. To provide further reassurance on this, we established separately that our indomethacin treatment protocol suppressed PGE2 levels in the fetal testis on e21.5 (Fig S1). However, fetal exposure to indomethacin during the MPW did not significantly alter intratesticular testosterone (ITT) levels at e17.5, in comparison to vehicle exposed animals (Fig 1B). Testosterone levels/androgen action during the MPW is reflected in AGD/AGI measurements from e21.5 onwards [4],[5]. In keeping with the data for e17.5 ITT, fetal indomethacin exposure during the MPW did not affect AGI at e21.5 (Fig. 2A), but did decrease fetal body weight of e21.5 male fetuses (Fig. 2B). Furthermore, exposure to indomethacin caused a significant decrease (27%) in fetal testis weight at e21.5 and this decrease occurred despite the fact that indomethacin treatment had ceased after e18.5 (Fig. 2C). If indomethacin treatment (0.8 mg.kg/day) was administered for a longer period (e15.5–e20.5), a similar decrease was found in testis weight (data not shown).

Affiliation:
MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, United Kingdom.

ABSTRACTRecent studies have established that masculinization of the male reproductive tract is programmed by androgens in a critical fetal 'masculinization programming window' (MPW). What is peculiar to androgen action during this period is, however, unknown. Studies from 20 years ago in mice implicated prostaglandin (PG)-mediation of androgen-induced masculinization, but this has never been followed up. We therefore investigated if PGs might mediate androgen effects in the MPW by exposing pregnant rats to indomethacin (which blocks PG production by inhibiting cyclooxygenase activity) during this period and then examining if androgen production or action (masculinization) was affected. Pregnant rats were treated with indomethacin (0.8 mg/kg/day; e15.5-e18.5) to encompass the MPW. Indomethacin exposure decreased fetal bodyweight (e21.5), testis weight (e21.5) and testicular PGE2 (e17.5, e21.5), but had no effect on intratesticular testosterone (ITT; e17.5) or anogenital index (AGI; e21.5). Postnatally, AGI, testis weight and blood testosterone were unaffected by indomethacin exposure and no cryptorchidism or hypospadias occurred. Penis length was normal in indomethacin-exposed animals at Pnd25 but was reduced by 26% (p<0.001) in adulthood, an effect that is unexplained. Our results demonstrate that indomethacin can effectively decrease intra-testicular PGE2 level. However, the resulting male phenotype does not support a role for PGs in mediating androgen-induced masculinization during the MPW in rats. The contrast with previous mouse studies is unexplained but may reflect a species difference.